Research On The Ultrasonic Lipid Microbubble Mediated By NGR And CPPs Targeting To Tumor Cells For SiRNA Delivery

We have known that the Cell penetrating peptides(CPPs) have a good function in penetrating the cell membrane. But also, there were two disadvantage factors limited its effects applied in our bodies. One of the factors was that the CPPs lacks specificity which made any cell membrane would be through in when they meet the CPPs. The other one was the CPPs could be degraded by enzymes easily in blood. Because of the two defects mentioned above, the CPPs must hide themselves functionally till they reach their target, and then they would be activated by stimuli in vivo or in vitro.As a new drug carrier, the Ultrasonic Lipid microbubble(ULM) not only enhanced the ultrasound image, but also worked on targeted therapy. The ULM had two main advantages when it working as a drug carrier. One was its ability of physical target and chemistry target. The process proceed with a located irradiation on the surface of gross tumor volume(GTV) with a certain intensity ultrasound. Then the microbubble ruptured in the targeted tumor tissue which caused by ultrasound-mediated microbubble cavitation, and the drugs carried by microbubble would be released to the GTV in order to accomplish the goals to release targeted drugs. The other one was the the ULM could improve the transfection efficiency. A lot of power was released at local instantly when the microbubble ruptured, which caused an invertible slight-damaged on the membrane of targeted cell. As a result, the membrane permeability increased which enhanced the gene drugs intake. Therefore, we can combine the advantage functions of Ultrasonic Lipid microbubble and CPPs, form a mutually complementary and beneficial pattern to ensure the gene drugs a high transfection efficiency.In this study, the main research content was building a multifunctional new gene carrier. Different from the gene carriers reported in existing literatures, in this study, the covalent attachment of CPPs and small-interfering RNA(siRNA) was packaged in the ULM, in order to hide the function of CPPS before it arrive at the target cell. Then gave a located irradiation on the GTV with a certain intensity ultrasound by which the gas in microbubble oscillated and expanded to rupture. On the one hand, the membrane permeability increased which enhanced the gene drugs intake. On the other hand, the CPPs would be exposed and activated. Present study mainly included the following 3 parts. The first part was the synthesis and characterized of NGR-PEG2000-DSPE and CPPs-siRNA. The second part was the preparation and characterized of CPPs-siRNA/NGR-ULM. The last part was cell evaluation in vitro. In the last part, we mainly investigated the CPPs-siRNA/NGR-ULM intakes and distribution in cell HT-1080, endosomal escaping and the condition of cell apoptosis. Based on those studies, we evaluated the tumor targeting of carrier and transfection efficiency in a tumor cells model level.Firstly, we synthesized the CPPs-siRNA by the disulfide bond formed in the oxidizing reaction between 5’ tip’s hydrosulfuryl on the ositive-sense strand of siRNA and the 5’ tip’s hydrosulfuryl on the CPPs, while, the products have been tested and verified by Matrix Assisted Laser Desorption Ionization Time of Flight(MALDI-TOF)-Mass Spectrometer. Then, the NGR-PEG2000-DSPE, the function materials, was prepared by the michael addition reaction, and also been tested and verified by MALDI-TOF-Mass Spectrometer. Lastly, the CPPs-siRNA/ULM and CPPs-siRNA/NGR-ULM were prepared by film dispersion-ultrasonic, repetitive freeze thaw and ultraphonic air-entrapping, and the physical and chemical properties have been characterized. Results shown that the sample was an Irregular polygonous particle(diameter was about 150 nm) and the entrapment efficiency was about 85%. The particles’ stability was good in 24 hours and could be irradiated with ultrasound at 0.6 mechanical index in 5 minutes while the releasing rate of CPPs-siRNA could up to above 80%.Our study evaluated the CPPs-siRNA/ULM and CPPs-siRNA/NGR-ULM in vitro cell level with HT-1080 cells, the model cell. Results shown that the combining capacity of CPPs-siRNA/NGR-ULM with HT-1080 cell, which express CD13 highly, was increased markedly after been embellished from NGR. With the laser scanning confocal microscope, we observed that beening activated by ultrasound, the CPPs-siRNA/NGR-ULM could escape the late endosome/lysosomes after entering in cell. Moreover, the target embellish gene brought in and ultrasound activation could enhance the gene silencing effect of CPPs-siRNA/NGR-ULM significantly, then shown us a remarkable apoptosis of tumour cell(about 54%). In ultrasound activated CPPs-siRNA/NGR-ULM group, the synergistic effect of, NGR, ULM and CPPs activated by ultrasound enhanced the uptake rate of HT-1080 cell and the inhibited effect in proliferation of HT-1080 cell.Based on those results, we came to following conclusion. The NGR cooperated with ultrasound activated CPPs acted on the ULM, a new multifunctional carrier, have shown a fairly ideal antineoplastic effect which open up a new way in the research of tumor targeting therapy.